Sequencing by Synthesis (SBS) has driven much of the “next generation” sequencing technology, allowing the field to approach the $100,000 Genome (1-4). With further improvements in nucleotide incorporation detection methods, SBS could be an engine that drives third-generation platforms leading to the reality of the “$1,000 Genome”. At the same time, since non-fluorescent detection approaches are likely to decrease the cost of obtaining data by avoiding expensive cameras and imaging tools, SBS also offers the possibility of high sensitivity, leading to both longer reads and permitting single molecule sequencing, thereby removing one of the most time-consuming and biased steps, the generation and amplification of DNA templates.
Some commercial platforms have been able to achieve direct single molecule sequencing but at the expense of accuracy (e.g., a single fluorescent tag in the case of the Helicoscope tSMS™ technology, 4 different fluorophores in Pacific Biosciences' SMRT sequencing approach, or illumination of 4 different fluors by enzyme-attached quantum dots in Life Technologies SMS system) (5-7). The shortcoming in all these approaches is that their dependence upon precise timing of a “virtual” pause between each nucleotide incorporation event, especially when registering the incorporation of more than a single base. This becomes particularly pronounced with homopolymeric runs of more than about 4 bases, which are often resolved by summing the fluorescent signals, rather than attempting to measure their timing (8, 9). The use of reversible terminators overcomes this obstacle by only allowing a single base to be incorporated prior to the detection step; only after subsequent cleavage of the terminating moiety on the nucleotide, can the next one be incorporated and identified (10-13). In the case of an already established system with fluorescently tagged nucleotide reversible terminators (NRTs), because each of the nucleotides has a separate fluorescent tag, all four can be added at the same time, reducing the number of rounds of incorporation 4-fold (14, 15). It is noteworthy that this strategy has also been shown to solve the accuracy problem for pyrosequencing, used by a Roche sequencing platform, which is not a single-molecule approach (16).